JP2010175355A - Automatic analyzer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enable a high-sensitive analysis being accompanied by a low noise occurrence and a low background to be carried out in an analytical method which employs a tracer, a labeled antibody, a labeled antigen or the like for binding a radioactive isotope, a fluorescent dye, a luminescent dye, an enzyme or the like as an antibody or an antigen bonding to a protein of an analysis object for each analysis item of proteins, in order to carry out a measurement analysis of a minute amount of matter such as a hormone, a tumor marker, an infection pathogen marker, an infection protective antibody or the like, wherein blood, blood serum, blood plasma or body fluid is used as a sample or an analyte. <P>SOLUTION: When carrying out an analysis measurement on the basis of an antigen-antibody reaction of the sample in an apparatus or the analytical method employing a fluorescence measuring method or a luminescence measuring method, nonspecific fluorescence or luminescence such as interference light or the like is effectively removed, or an operation of detecting or measuring them is set aside, regardless of concentration degrees of matters contained in the sample or aspects of samples, thereby carrying out the high-sensitive analytical measurement. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  In the present invention, when blood, serum or plasma is used as a sample or specimen and analysis of a trace substance in blood is performed using an antigen-antibody reaction, a phosphor that binds to an antigen or an antibody for the purpose of knowing the presence or absence of the reaction or the amount of reaction. The present invention relates to an antigen or antibody analysis method using a luminescent material.

  When measuring and analyzing trace substances such as hormones, tumor markers, infectious disease pathogen markers, and infectious antibodies using blood, serum, plasma, or body fluid as a sample or specimen, binding based on antigen-antibody binding reaction for each protein analysis item Thus, a method for qualitatively or quantitatively detecting a protein or the like in a sample or a specimen is generally used. In this case, the antibody or antigen that binds to the protein to be analyzed is bound to a radioisotope, fluorescent dye, luminescent dye, enzyme, rare earth complex, metal ion, etc., and is called a labeled antigen, labeled antibody, or tracer. Yes.

  Similarly, when measuring or analyzing DNA or RNA such as pathogens and drug metabolism markers in a sample or specimen, the DNA or RNA strand complementary to the DNA or RNA of the analyte is hybridized to detect the analyte. To do. At this time, the complementary DNA strand or RNA strand is directly or indirectly labeled with a phosphor or a light emitter.

  The immunological analysis method is a specific measurement method of a biological substance based on the fact that an antigen and an antibody that binds to the antigen generate an antigen-antibody bond. Depending on the measurement principle, there are an in-solution precipitation reaction method, a carrier agglutination reaction method, and a labeled antibody method. The in-solution precipitation reaction method is a method for optically measuring and quantifying an aggregate produced by antigen-antibody binding in a solution, and an immunoturbidimetric method, an immunotrophic method, and the like are known. The agglutination reaction method using a carrier is a method in which a carrier such as latex in which an antibody is solid-phased and a sample solution (antigen) are bound to an antigen-antibody, and the resulting agglutination of the carrier is optically or imageically measured and quantified. It measures the increase in apparent particle size or decrease in transmitted light by the turbidity method, latex specific wax method, particle count, image processing, etc. The labeled antibody method is a method in which antigen-antibody binding is performed using antibodies labeled with various labeling substances, and only the component that has reacted with the labeled antibody is measured. Radioimunoassay, enzyme immunoassay, fluorescent immunoassay, Chemiluminescence immunoassay, electrochemiluminescence immunoassay and the like are known.

The problems pointed out by immunological analysis are listed below.
1. Effects of heterophile antibodies: In systems where monoclonal antibodies are used, if HAMA (human anti-mouse antibody) is present in the sample, the labeled antibody and the solid phase antibody are cross-linked to cause false positives.
2. Non-specific reaction with blocking agent, etc .: The blocking agent or protective protein contained in the reagent reacts with the substance in the sample, causing false positives.
3. Nonspecific reaction with labeling enzyme: Background increases if a substance that binds to the labeling enzyme is present in the sample.
4). Prozone phenomenon or high dose huck: False negative when the antigen concentration is above the measurement range or the labeled antibody concentration is significantly above the instrument's measurement range. In the antigen excess region, the antigen-antibody complex becomes soluble and may show an abnormally low value.
5). Influence of hemolysis: If the sample is very hemolyzed, it will affect the color of the judgment, making the judgment difficult.
6). If the sample is stool, urine, etc .: Use a fresh sample. Samples containing strong cloudy urine or blood cells are not suitable.
7). Influence of sample properties: When the viscosity of the sample, especially when the protein concentration is high (M protein, cryoglobulin positive sample), the reaction rate is slow and false negative. Retest with diluted sample. Nonspecific scattering may occur in the presence of chyle serum or immune complexes.
8). False positive and false negative due to cross-reaction: found in systems that use recombinant antigens. In FSH, TSH, and LH, false positives occur due to cross-reactions.
9. Effects of drugs used (atropine, caffeine, acetamidophenol, acetylsalicylic acid, ascorbic acid, etc.): The urinary hCG measurement system is affected and becomes false positive.
10. Measurement sensitivity difference between reagents: There are cases in which a positive reaction appears after a certain time, although it is negative at the specified judgment time.

  Many years have passed since Barson and Yarrow completed the radioimmunoassay by labeling radioisotopes with antigens and antibodies. Due to its high sensitivity and high specificity, it is still used as a useful method. On the other hand, development improvements such as enzyme immunoassay, fluorescence immunoassay, and luminescence immunoassay have been made during this period.

  The enzyme immunoassay is a method in which an enzyme is labeled with an antigen or an antibody instead of a radioisotope, and measurement with high sensitivity is possible due to the high catalytic activity of the enzyme on the substrate.

  A time-resolved fluorescence measurement method was developed to measure the fluorescence of this substance after the time when the fluorescence generated from the reaction vessel or the like has been quenched by irradiating the antigen-antibody reaction final product with pulses of excitation light. In recent years, a europium complex or a samarium complex having a relatively long fluorescence quenching time or one that does not require a sensitizer has been developed, and this technique has become more effective.

  In the luminescence immunoassay method, a method of measuring the amount of luminescence by labeling isoluminol or acridinium ester, a combination of peroxidase and luminol using a labeling substance that is an enzyme but can produce a luminescence reaction on the substrate, or In addition, a measurement system based on a combination of a substrate such as AMPPD that emits light when a phosphate group is removed and alkaline phosphatase, or a combination that uses luciferase as a labeling substance and luciferin as a substrate has been constructed.

  In labeled antigen, labeled antibody, labeled DNA, labeled RNA, tracer, etc., a fluorescent substance or a luminescent substance is bound to a labeled substance as a single substance, but there are many for the purpose of increasing the amount of fluorescence or luminescence. Attempts have been made to bind the phosphor or luminescent material to one label. Multiple phosphors or phosphors can be combined as a complex, or as a complex with bovine serum albumin, streptavidin, or other proteins, or bound to the surface of microparticles, and this can be bound as a label to the label. Made. As fine particles, gelatin, latex, or polystyrene particles having a diameter of about 2 micrometers (μm) are generally used, and they are used as carriers for phosphors or luminescent materials. In recent years, particles having a diameter of about 100 nanometers (nm) have been developed. In many cases, the phosphor or phosphor is bound to the carrier or the solid phase is bound by electrical coupling after mixing the microparticles and the phosphor or phosphor in a buffer solution.

  The reaction surface used as a place for antigen-antibody reaction or gene detection method is generally plastic, glass plate or particles, and magnetic particles containing iron oxide are also used. Also, filters, fiber weaves, multilayer papers, and porous membranes may be used to increase the solid surface area.

  When solids with reaction surfaces are particles, they are put in a reaction vessel and mixed with a sample, specimen, or reagent. These containers contain transparent ores such as quartz and their artificial stones, glass, or polycarbonate. For example, a compound cell such as a material, a polystyrene material or a polyvinyl material or a microtiter plate may be used.

  As a method for measuring immunity / serum analysis items in clinical laboratory tests, there are many techniques for detecting a reaction using an antibody or antigen having a fluorescent substance or a luminescent substance or an antigen as a label using an antigen-antibody reaction. At this time, in the case of a fluorescent substance such as a dye, a fluorescent dye or a phosphorescent dye, or a luminescent substance, an antigen-antibody reaction is detected even if these substances are directly labeled with an antibody or an antigen. However, if a substance to be labeled is bound to a plurality of substances to form a complex of a phosphor or a luminescent material and a carrier, and this is bound to an antigen or an antibody to produce a labeled antibody or antigen, 1 The number of fluorophores or labels that bind to molecular antigens or antibodies increases, and a stronger signal can be generated. When using proteins such as bovine serum albumin and keyhole limpet hemocyanin, plastic particles such as polystyrene and polypropylene, other polymers such as ferrite, silica, and gelatin, or chain carbon compounds such as polyvinyl alcohol, these are labeled carriers. It hits.

  A plurality of phosphors or phosphors can be encapsulated in liposomes, lipid membranes or hollow plastics, thereby amplifying the signal. In this case, liposome, lipid membrane or hollow plastic corresponds to the carrier.

  This carrier forms a complex with a phosphor or a light emitter and is used as a label. The label may form huge aggregates during storage. In addition, the formation of the complex significantly decreases the thermal stability and easily adsorbs to the reaction vessel or the reaction solid phase body, which increases the non-specific background. Similarly, quenching or quenching in which coloring or luminescence or fluorescence of the substance is reduced occurs from the properties of the carrier and the labeling substance and from the components contained in the solution. Furthermore, when the antigen and antibody are labeled, the labeled antibody molecule is large, so that the antigen-antibody reaction is significantly reduced.

  In an antigen-antibody reaction by a fluorescence method or a luminescence method, a fluorescent substance, a luminescent substance, or an enzyme that is bound to a labeled antibody or a labeled antigen is not bound to an antibody or an antigen in a one-to-one relationship, In general, a luminescent substance or an enzyme is bound to an antibody or a single antigen molecule. As a result, the signal is amplified and taken out.

  A plurality of fluorescent substances, luminescent substances, or enzymes are bound to other proteins or supports, and these proteins or supports are used as carriers such as fluorescent substances. It is important that this carrier has the following requirements:

1. The carrier preferably has a low molecular weight but a large surface area, and a specific gravity close to the buffer used.
2. It is preferable that the carrier is easy to solidify a fluorescent substance and the like and does not reduce the fluorescence emission performance of the fluorescent substance.
3. After labeling on a carrier such as a fluorescent substance, the carrier should preferably be difficult to adsorb to the reaction vessel or other particles.
4). The carrier should be fine and of a size that does not optically block the excitation light. Similarly, the carrier should not inhibit fluorescence or light emission from being detected by a photomultiplier tube or the like. If there is an object shorter than the wavelength, it will not be blocked by this light. If the carrier is shorter than the wavelength of light used, the light passes through it without being attenuated even if it does not pass through the carrier. Further, there is no scattering by the carrier, and the generation of noise is minimized.
5). Further, the carrier is preferably colorless and transparent and does not hinder the passage of excitation light. In addition, it is preferable that non-specific fluorescence and luminescence are not generated by excitation light irradiation.
6). The carrier is preferably highly hydrophilic and highly dispersible. Similarly, it is preferable that hydrophilicity and dispersibility are easily maintained after labeling with a fluorescent substance or the like.

  The homogeneous measurement method (homogeneous method) has been performed as a method in which the measurement is always performed in a solution state. However, as a highly sensitive measurement is required, a free labeled antibody that does not participate in the reaction by reacting an antigen with an antibody ( Alternatively, a method of measuring an antigen-antibody complex after separating (antigen) by washing operation (B / F separation), that is, a heterogeneous measurement method (heterogeneous method) is widely used. Today, the FRET method, which detects the fluorescence of the second substance by excitation by the combination of the fluorescent dye and its quencher (quenching dye), or energy transfer from the first fluorescent substance to the second fluorescent substance, oxygen channeling With the development of the LOCI method that applies, and the gold particle method that measures different color development due to the proximity and aggregation of gold colloid particles, a homogeneous method with high sensitivity has become possible.

  In Patent Document 1, a first metal particle and a second metal particle are arranged on a substrate at an interval that does not cause mutual interaction, and are coupled to the first metal particle and the first metal particle. The second metal particles having the first substance and bonded to the second substance are supplied to the substrate, and the first metal particle and the second substance are connected to the substrate through the bond between the first substance and the second substance. A method of observing the binding between the first substance and the second substance by binding metal particles and causing mutual interaction with each other was shown. Patent Document 2 discloses an analyte dye complex with an analyte (antigen) bound with a non-fluorescent dye, an antibody against the analyte dye complex, and an antibody that recognizes both the analyte and the dye as an epitope; A method for quantifying the amount of the analyte by measuring the fluorescence intensity based on the antigen-antibody reaction between the analyte dye complex and the antibody was shown.

  In either method, special metal particles, substrates, dyes, or antibodies that recognize two epitopes need to be prepared, and a special technique is required to create a system. To do so, it is far from practical use and requires further development.

JP 2003-14765 A JP 2007-171213 A

  As the above-mentioned conventional technique requires a uniform measurement method (homogeneous method) that realizes high-sensitivity measurement as a method in which measurement is always performed in a solution state, a combination of a fluorescent dye and its quencher (quenching dye) FRET method that detects the fluorescence of the second substance by excitation by energy transfer from one fluorescent substance to the second fluorescent substance, LOCI method that applies oxygen channeling, and different color development due to proximity and aggregation of colloidal gold particles With the development of the gold particle method, etc., a highly sensitive homogeneous method has become possible.

  Factors that make analysis based on antigen-antibody reaction unstable are the effects of heterophilic antibodies, false positives due to non-specific reactions with blocking agents, background increase due to non-specific reactions with labeled enzymes, prozone phenomenon or high False negative in the case of dose huck, false negative or false positive due to the influence of the properties of samples such as high protein sample, chyle serum, false positive and false negative due to cross reaction when using recombinant antigen, crossover in FSH, TSH, LH There are false positives due to the reaction, instability of determination results due to the difference in measurement sensitivity between reagents, and the like.

  In addition, the fluorescence or light emission indicating interference is inherently measured and measured as if the measurement object is present in the sample, even though the measurement object is not included in the sample, resulting in an erroneous determination. May also occur, and may further reduce the minimum detection limit of the measuring device.

  The object of the present invention is to use interference light when performing analytical measurement based on an antigen-antibody reaction of a sample in an analysis method or apparatus based on a fluorescence measurement method or a luminescence measurement method, regardless of the nature of the sample or the concentration of a substance contained in the sample. It eliminates or effectively eliminates non-specific fluorescence or luminescence, or eliminates it effectively, and attempts to perform highly sensitive analytical measurement.

  Another object of the present invention is to determine the concentration of an analyte contained in a sample when an analytical method based on an antigen-antibody reaction of the sample is performed in an analysis method or apparatus based on a fluorescence measurement method or a luminescence measurement method. It eliminates or effectively eliminates the detection or measurement of specific fluorescence or luminescence, and promptly performs analytical measurement.

  Furthermore, the object of the present invention is to analyze the properties of the sample or the concentration of the substance other than the analyte contained in the sample when performing an analytical measurement based on the antigen-antibody reaction of the sample in an analysis method or apparatus based on a fluorescence measurement method or a luminescence measurement method. The concentration of the analyte is rapidly and highly sensitively measured based on the antigen-antibody reaction.

  In the present invention, when performing an analytical measurement based on an antigen-antibody reaction of a sample in an analysis method or apparatus based on a fluorescence measurement method or a luminescence measurement method, interference light or the like is not affected regardless of the property of the sample or the concentration of the substance contained in the sample. It is possible to eliminate or effectively exclude the detection or measurement of specific fluorescence or luminescence and perform highly sensitive analytical measurement.

  Further, in the present invention, when an analytical measurement based on an antigen-antibody reaction of a sample is performed in an analysis method or apparatus based on a fluorescence measurement method or a luminescence measurement method, the concentration of an analyte contained in the sample is determined as non-specificity such as interference light. This eliminates or effectively eliminates the detection or measurement of typical fluorescence or luminescence, and enables rapid analysis and measurement.

  Furthermore, in the present invention, when an analytical measurement based on an antigen-antibody reaction of a sample is performed in an analysis method or apparatus based on a fluorescence measurement method or a luminescence measurement method, the properties of the sample or a substance concentration other than the analyte contained in the sample High and low can be estimated effectively, and the analyte concentration can be measured quickly and with high sensitivity based on the antigen-antibody reaction.

(1) According to the present invention, when an analytical measurement based on an antigen-antibody reaction of a sample is performed in an analysis method or apparatus based on a fluorescence measurement method or a luminescence measurement method, the concentration of an analyte contained in the sample is changed to interference light or the like. It is possible to eliminate or effectively exclude non-specific fluorescence or luminescence, and to quickly perform analytical measurement.
(2) Furthermore, according to the present invention, when performing an analytical measurement based on an antigen-antibody reaction of a sample in an analysis method or apparatus based on a fluorescence measurement method or a luminescence measurement method, other than the properties of the sample or the analyte contained in the sample The substance concentration can be effectively estimated and the analyte concentration can be measured quickly and with high sensitivity based on the antigen-antibody reaction.

An example of an overview of the analyzer. An example of an antigen-antibody reaction. FIG. 3 is a schematic diagram when a reaction solution is collected from a reaction container at a specified time and light emission or fluorescence measurement is sequentially advanced. The reference schematic diagram in the case of progressing light emission or fluorescence measurement for every specified time, without collecting the reaction liquid in a reaction container. Example of reaction time curve (1). Example of reaction time curve when using low value sample (2). Example of reaction time curve when high value sample is used (3).

  Embodiments of the present invention will be described below with reference to the drawings.

  A configuration example of the immune analysis unit 103 will be described with reference to FIG. In FIG. 1, a plurality of reagent containers 201 containing reagent solutions corresponding to analysis items that can be analyzed by the immunological analysis unit are arranged on a reagent disk 202 that can be rotated as a reagent positioning device. The reaction disk 203 maintained at a constant temperature can be rotated, and there are a plurality of reaction positions along the circumference on the reaction disk 203, and the reaction container 205 from the reaction container storage position 219 is stored therein. The reaction disk 203 moves the reaction container 205 from the reaction container installation position 204 to the sample discharge position 221, the reagent addition position 222, and the reaction liquid suction position 212 by a rotating operation. The sample dispensing pipetter 206 can move the coupling tube coupling the disposable dispensing tip 210 in the horizontal direction from the upper part of the sample suction position 207 to the upper part of the sample discharge position 221 and can be moved up and down at each position. It has become. Prior to the suction of the sample, the disposable dispensing tip 210 is attached to the tip of the tip coupling tube of the sample dispensing pipetter 206 at the tip coupling position 218.

  The reagent dispensing pipettor 208 can move from the upper part of the reagent suction position 209 on the reagent disk 202 to the upper part of the reagent addition position 222, and can move up and down at each position. The sipper 211 can move between the upper part of the reaction liquid suction position 212, the upper part of the buffer liquid suction position 213, and the upper part of the cleaning liquid suction position 214 for the flow cell, and can also move up and down at each position. Further, the shipper 211 has a function of sending the reaction solution to the flow cell in the detection unit 215 through the tube. The tip and reaction vessel transfer mechanism 216 that can move the gripping part in the x and y directions has the disposable dispensing tip 210 from the tip storage position 217 to the tip coupling position 218 and the disposable reaction vessel 205 to the reaction vessel storage position 219. To the reaction vessel installation position 204. In the reagent dispensing pipettor 208 and the sipper 211, the outer wall of the nozzle is washed with water at each corresponding washing position.

  Next, the flow of processing in the immune analysis unit 103 will be described. First, the chip and reaction container transfer mechanism 216 transfers the disposable dispensing chip 210 to the chip coupling position 218 and then the reaction container 205 to the reaction container installation position 204. The rack 107 holding the sample container 108 is conveyed on the subline 113 so that the sample container 108 containing the sample to be analyzed is positioned at the sample suction position 207. At the same time, the reagent disk 202 rotates so that the reagent container 201 containing the reagent used for the analysis is positioned at the reagent suction position 209. At the same time, the reagent dispensing pipettor 208 moves to the upper part of the reagent suction position 209. At the reagent aspirating position 209, the reagent dispensing pipettor 208 descends and aspirates the reagent into the pipette nozzle. Next, the reagent dispensing pipettor 208 moves up and moves to the nozzle cleaning position. When the pipette nozzle reaches the upper part of the nozzle cleaning position, cleaning water blows out from the cleaning tank, and the tip of the pipette nozzle is cleaned.

  On the other hand, the sample dispensing pipetter 206 moves the dispensing tip 210 to the upper part of the sample suction position 207, descends into the sample container 108 on the rack 107, and sucks a predetermined amount of sample. After the sample is aspirated, the dispensing tip moves up and moves to the sample discharge position 221. Then, the dispensing tip is lowered, and the sample that has been sucked and held in the dispensing tip is discharged into the reaction container 205. After the sample is discharged, the dispensing tip is lifted by the sample dispensing pipetter 206 and moved to the tip disposal position 220. At the tip disposal position 220, the sample dispensing pipetter 206 removes the disposable dispensing tip 210 from the coupling tube and discards it.

  After a predetermined time required for the reaction has elapsed, the sipper 211 moves the suction nozzle to the upper part of the buffer solution suction position 213, lowers the nozzle, and sucks the buffer solution through the nozzle toward the flow cell. Thereafter, the tip of the nozzle of the sipper 211 is cleaned at the nozzle cleaning position.

  Next, the reaction disk 203 transfers the reaction container 205 to the reaction liquid suction position 212. The sipper 211 sucks the reaction liquid through the nozzle toward the flow cell at the reaction liquid suction position 212. After sucking the reaction solution, the sipper 211 moves the nozzle to the buffer solution suction position 213 and sucks the buffer solution. The sucked buffer solution and reaction solution are sent to the flow cell in the detection unit 215 through the tube, and measurement is performed. Then, the sipper 211 moves the nozzle to the cleaning liquid suction position 214, sucks the cleaning liquid for the flow cell, and cleans the inside of the flow cell in the detection unit 215 with the cleaning liquid.

  Next, a configuration example of the analysis unit 104 will be described with reference to FIG. In FIG. 2, the biochemical analysis unit 104 includes a reagent supply system including reagent disks 301A and 310B for holding a large number of reagent containers 310 and reagent dispensing pipettors 302A and 302B, and a sample supply system including a sample dispensing pipetter 303. And a reaction unit including a reaction disk 305 for holding a large number of reaction vessels 304, and a measurement system including a multi-wavelength photometer 306 and an analog / digital converter 307.

  In FIG. 2, the rack 107 holding the sample container 108 is transported from the transport unit 102 to the sample suction position 308 on the subline 115. The sample dispensing pipetter 303 sucks a predetermined amount of the sample in the sample container 108 into the pipette nozzle 401 and discharges the sample into the reaction container 304.

  The reaction container 304 into which the sample liquid has been discharged and dispensed is moved to the first reagent addition position by the rotation of the reaction disk 305 that is kept warm by the thermostat 309. At this time, the reagent disk 301A is also moved by the rotating operation so that the reagent container 310 corresponding to the analysis item of the sample coming to the reagent addition position is positioned at the reagent suction position.

  Then, the predetermined first reagent sucked by the pipette nozzle of the reagent dispensing pipettor 302A is added to the reaction container 304 moved to the first reagent addition position. The reaction vessel 304 after the addition of the first reagent is moved to the position of the stirring device 311 and the first stirring is performed. In the case of an analysis item that requires the addition of the second reagent, the second reagent is further added by the reagent dispensing pipette 302B, and the contents are stirred.

  The reaction container 304 containing the reaction liquid in which the sample and the reagent are mixed is transferred so as to cross the light beam from the light source, and the light transmitted through the reaction container enters the multi-wavelength photometer 306. Then, the absorbance of the reaction liquid that is the contents of the reaction vessel 304 is detected by the multiwavelength photometer 306. The detected absorbance signal is supplied to the control unit 312 configured by a computer via an analog / digital (A / D) converter 307 and an interface, and is converted into the concentration of the analysis item to be measured in the sample. The reaction vessel 304 that has been subjected to the analytical measurement is moved to the position of the reaction vessel cleaning mechanism (not shown), and after the reaction liquid in the reaction vessel is discharged by the reaction vessel cleaning mechanism, the reaction vessel 304 is washed with water for the next analysis. To be served.

  A technique that enables continuous measurement or multiple measurements is described below.

  FIG. 3 shows that reaction solutions are sequentially collected from one reaction vessel and used to measure luminescence values. At the time of measuring each luminescence amount, a certain amount of reaction solution is collected and luminescence measurement is carried out. However, the reaction solution remaining in the container is kept in the incubator until the final luminescence measurement is continued. When collecting the reaction solution, the container may be carried out in the incubator, or the solution may be collected by taking it out of the incubator. When the composition of the reaction solution during the incubation is uneven, it is desirable to collect the reaction solution after stirring and mixing the reaction solution.

  FIG. 4 shows an example in which luminescence measurement is performed over time or every specified time without collecting the reaction solution in the reaction vessel. The container is provided with an opening through which a sample or reagent is dispensed. On the other hand, a part or the whole of the container has a part where an antibody, antigen or other substance that captures the measurement target is solid-phased, and the reaction proceeds at this part. Alternatively, when there are solid phase particles or the like in the reaction solution and the reaction proceeds on the particles, it is not always necessary to provide a solid phase portion in the container. FIG. 4 shows that the solid phase site is separated from the reaction solution by tilting the container during luminescence measurement, and fluorescence measurement is performed at this location. There may be unreacted fluorescent labeling substances such as fluorescent dye-labeled antibodies that have not reached the reaction in the solid phase in the reaction solution, and when performing luminescence or fluorescence measurement using a liquid containing these, from the unreacted labeling substance The background at the time of measurement increases due to the luminescence or fluorescence generated. For this reason, it may become an obstacle when trying to realize a measurement with higher sensitivity, and it is desirable to take a method of removing these backgrounds as much as possible. The reaction curve can be obtained by repeatedly performing this luminescence measurement and returning the container to the original state and attaching the solid phase portion to the reaction solution to continue the reaction. The measurement object shown in Example 3 can be obtained more quickly and accurately. You can analyze things.

  It is desirable to perform real-time measurement if the number of luminescence measurements is large, but it is more reliable to obtain a measurement value in the middle of one measurement in addition to the final measurement more quickly, more accurately, and without retesting. Analysis is made and it can be seen that it has a great advantage for disease diagnosis.

  An example of a signal curve over time of the antigen-antibody reaction is shown in FIG. In general, the luminescence value or signal increases with the progress of the reaction, but the increase in the luminescence value becomes small after a certain period of time. At this point, the reaction is terminated, and the luminescence value is measured. From this value, it is determined that the antigen or antibody in the subject is contained or not. The reference value or boundary value at this time is referred to as a cut-off value, and if a light emission value higher than this is obtained, it is determined to be positive, and if it is lower, it is determined to be negative (qualitative measurement). The vicinity of this cutoff value is set as a gray zone, and here, determination may be impossible and re-measurement may be requested. In some cases, the amount of the measurement object is quantitatively described by comparing the luminescence value with a calibration curve (quantitative measurement). In the example of FIG. 5, the antigen-antibody reaction is completed at a reaction time of 15 minutes and the luminescence value is measured, and a qualitative measurement of whether the cut-off value is exceeded or below, or how much the measurement object is measured by comparing the luminescence value with a calibration curve. Get readings that are included. A is determined to be negative without exceeding the cutoff value, and B, C, and D are determined to be positive. Alternatively, readings are obtained from a calibration curve and used for diagnosis.

  Compared to the conventional method of completing the reaction for 15 minutes or at the end of the reaction and collecting the luminescence value once, the advantage of obtaining the luminescence value multiple times over time is illustrated. By measuring the luminescence value at the first time or at 5 minutes, it can be seen that the C and D samples exceed the cutoff value, and the C and D samples are determined to be positive. On the other hand, it is desirable that the light emission value measurement or photometry can be performed continuously in the samples A and B, but the advantage is enhanced by performing the reaction process twice or a plurality of times.

  The case where a luminescence value is obtained with time for a negative or weakly positive sample is shown below. At 15 minutes, E is negative and F and G are positive. In G, it was determined to be positive even at 10 minutes, and early results could be determined without waiting for the final 15 minutes. In F, it can be determined to be positive at 15 minutes, but it can be estimated that the measurement at 5 minutes and 10 minutes will be positive at 15 minutes, and an early prediction can be made. Further, the result at 15 minutes can be backed up from the results at 5 minutes and 10 minutes, and the final determination result is more reliable.

  Next, FIG. 6 shows a case where a relatively large amount of measurement object is contained. All of H, I, and G were determined to be positive at 5 minutes, and the determination results could be known early after the start of the test. This result can be communicated to doctors more quickly. For example, if a patient suffers from a heart disease and needs immediate treatment such as a heart infarction, the chances of ending the life with more accurate medical treatment are increased. .

  In antigen-antibody reaction or its analyzer, there are many measurement objects called zone phenomenon, pro-zone, post-zone or high dose huck, or there are a lot of labels such as fluorescent dyes. In some cases, such as when the balance between the ratio of the antibody and the labeled antibody is poor, there is a possibility that it may be determined to be negative in an extreme case where it shows a small luminescence value despite the fact that there are a large amount of measurement objects in the sample. In such a case, it is possible to make a positive determination by knowing the luminescence value at the initial stage of the reaction, and the luminescence value here may more accurately reflect the amount of the measurement object than at the time of the final measurement. In FIG. 7, at the final measurement at 15 minutes, the luminescence measurement value can be read as H = J> I, and also from the calibration curve, H = J> I and the sample of H can be measured more than the sample of I. It is determined that the sample contains a high value. However, when the luminescence value at 5 minutes or 10 minutes was observed, it was read that J> I> H, and it was found that the most measurement object was included in the sample of J.

103 Immunoassay unit 201 Reagent container 202 Reagent disk 203 Reaction disk 205 Reaction container 206 Sample dispensing pipettor 208 Reagent dispensing pipettor 210 Dispensing tip 211 Shipper

Claims (5)

In an automatic analyzer comprising a reaction vessel for directly or indirectly binding a luminescent label to a measurement object and a measurement mechanism for detecting light emitted from the measurement object to which the luminescent label is bound,
An automatic analyzer comprising a moving mechanism for moving the measurement object to which the luminescent label in the reaction vessel is bound to the measurement mechanism a plurality of times in time series. The automatic analyzer according to claim 1, wherein
An automatic analyzer comprising a calculation mechanism for calculating an antigen-antibody reaction over time based on a result of time series measurement by the measurement mechanism. The automatic analyzer according to claim 1, wherein
An antigen-antibody conjugate that binds an antigen, antibody, or label to be labeled with a fluorescent substance or luminescent substance on a carrier is generated, and an antigen-antibody-bound fluorescent substance or luminescent substance that is not on the carrier in the reaction vessel An automatic analyzer comprising a removal mechanism for removing water. In the automatic analyzer in any one of Claims 1-3,
The automatic analyzer is characterized in that the moving mechanism collects the reaction solution in the reaction vessel part by time. In the automatic analyzer in any one of Claims 1-3,
The amount of antigen-antibody reaction can be obtained by assembling a part of the carrier in the reaction vessel or on the surface where antigen-antibody binding is generated, and changing the concentration of the phosphor or phosphor in the single assembly and the other reaction solution. An automatic analyzer characterized in that the antigen-antibody reaction is quantitatively measured sequentially by quantitatively measuring and detecting this multiple times from the start of the reaction.

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